Cell-based assays are increasingly gaining in popularity in the pharmaceutical industry due to their high physiological relevance. Additional advantages include their ability to predict compound usefulness, evaluate molecular interactions, identify toxicity, distinguish cell type-specific drug effects, and determine drug penetration. Cell-based assays are relevant throughout the drug discovery pipeline, because they are capable of providing data from target characterization and validation to lead identification (primary and secondary screening) to terminal stages of toxicology. Current industry trends of performing drug screening with cell context demand easily monitored, non-invasive reporters. This fundamental approach increases efficiency, reduces costs, and results in shorter time to market for new drugs. In order to fail compounds early, information-rich data for accurate early-stage decision making is required. Such data may be derived by screening compounds in context, that is, by screening in relevant living systems, rather than with classical biochemical assays. These screening systems often incorporate sophisticated imaging platforms, such as high-content screening (HCS) workstations. The industrialization of fluorescent microscopy has led to the development of these high-throughput imaging platforms capable of HCS. When coupled with fluorescent reporter technology, HCS has provided information-rich drug screens, as well as access to novel types of drug targets.
While suitable for analysis of cell surfaces and permeabilized cells, fluorescently-labeled antibodies have few practical applications for intracellular imaging in living cells, due to their inherent inability to penetrate to their targets, which has given rise to development of cell-permeable small molecule organic fluorochromes. Certain ones of these fluorochromes naturally sequester inside-specific organelles, based upon biophysical or biochemical properties favoring that distribution. Acceptable small molecule organic probes for cell imaging and analysis need to be minimally perturbing, versatile, stable, easy-to-use, and easy to detect using non-invasive imaging equipment. A problem with the classical organic probes from a histological standpoint is that many of them require cofactors or, by requiring fixation or staining, report only on the static condition of a dead cell. The required additional steps may be time consuming and expensive and, in the case of fixing and staining, may lack biological relevance. In the context of the analyses described above, an organic probe must be able to report upon events in living cells and in real time. Simplicity is of key importance, especially in the context of drug screening.
While various organic fluorochromes have been developed in the past for live cell analysis, typically they were devised using unperturbed cells. Thus, such organic fluorochromes statically report upon organelle spatial position within cells without the ability to monitor dynamic changes. For instance, several U.S. patent documents (U.S. Pat. Nos. 5,338,854, 5,459,268, 5,686,261, 5,869,689, 6,004,536, 6,140,500 and 6,291,203 B1, as well as US Patent Applications 2005/0054006 and 2007/0111251 A1, (foregoing patents and patent applications incorporated herein by reference) disclose organic fluorochromes which are described as useful for visualizing membranes, mitochondria, nuclei and/or acidic organelles. Additional examples of various fluorochromes and their application in biological imaging may be found in the published literature (see, for example, Pagano et al, 1989; Pagano et al, 1991; Deng et al, 1995; Poot et al, 1996; Diwu et al, 1999; Rutledge et al, 2000; Lee et al, 2003; Bassøe et al, 2003; Rosania et al, 2003, Li et al 2007; Boldyrev et al, 2007; Nadrigny et al, 2007). These dyes have been created using a number of fluorophores, most commonly dipyrrometheneboron difluoride (BODIPY), cyanine, carbocyanine, styryl and diaminoxanthene core structures. Typical emission maxima for these organic fluorophores span from 430 to 620 nm. Many of these dyes display certain suboptimal properties, such as a propensity to photo-bleach, metachromasy and even a tendency to photo-convert to different emission maxima upon brief exposure to broad-band illumination.
JC-1 dye is notable in its ability to monitor the dynamics of mitochondrial membrane potential changes and consequently the energetic state of cells (Smiley et al, 1991). Unfortunately, with respect to lysosomes and lysosome-like vesticles (autophagosomes, autophagolysosomes), there has been a notable lack of probes that have the ability to track organelle dynamics arising from drug treatment or intracellular membrane trafficking anomalies.
Spectral Problems Associated With Previously Devised Organic Fluorochromes for Analysis of Lysosomes
Fluorescence co-localization imaging is a powerful method for exploring the targeting of molecules to intracellular compartments and for screening of their associations and interactions. In these kinds of experiments, distinct fluorochromes and/or fluorescent proteins of interest are imaged as spectrally separated detection channels. The fluorescence intensity in each channel is ideally dominated by spatial and concentration information derived from one fluorophore only. Many commercially available organic fluorophores for subcellular analysis are disadvantaged in displaying suboptimal properties relating to these types of applications.
Lysotracker Red DND-99 (Invitrogen, Carlsbad, Calif.) contains a BODIPY fluorophore in the form of a conjugated multi-pyrrole ring structure and also contains a weakly basic amine that causes the fluorochrome to accumulate selectively in acidic compartments, exhibiting red fluorescence upon appropriate illumination (excitation: 577 nm, emission: 590 nm) (Freundt et al, 2007). Lysotracker Red is structurally related to Lysotracker Green but the former has an additional pyrrole ring in conjugation with the primary structure, which produces a longer wavelength emission. Lysotracker Red has commonly been used in multi-color imaging studies as a lysosomal marker to determine intracellular localization of GFP-tagged proteins by fluorescence or confocal microscopy. Excitation of the red-emitting molecule with broad-band illumination induces molecular changes, however, thus rendering its photochemical properties similar to those of Lysotracker Green. The similarities between the spectra of Lysotracker Green and converted Lysotracker Red suggest that the third pyrrole ring is taken out of conjugation during the photo-conversion process, leading to a shorter wavelength dye emission. Thus, Lysotracker Red staining for epifluorescence or confocal microscopy, in conjunction with visualization of GFP or FITC, leads to spurious results due to photo-conversion of the fluorophore (Freundt et al, 2007).
Acridine orange (Sigma-Aldrich, Saint Louis, Mo. and other sources) has also been used extensively as a fluorescent probe of lysosomes and other acidic subcellular compartments. Acridine orange's metachromasy results, however, in the concomitant emission of green and red fluorescence from stained cells and tissue (Nadrigny et al, 2007). Evanescent-field imaging with spectral fluorescence detection, as well as fluorescence lifetime imaging microscopy demonstrate that green fluorescent acridine orange monomers inevitably coexist with red fluorescing acridine orange dimers in labeled cells. The green monomer emission spectrally overlaps with that of GFP and produces a false apparent co-localization on dual-color images. Due to its complicated photochemistry and interaction with cellular constituents, acridine orange is a particularly problematic label for multi-color fluorescence imaging, both for dual-band and spectral detection. Extreme caution is required, therefore, when deriving quantitative co-localization information from images of GFP-tagged proteins in cells co-labeled with acridine orange.
Cationic amphiphilic drug tracers have been disclosed. See, for example, acridine-propranolol disclosed in Dickens et al., J Mol Cell Cardiol 34:129-137 (2002); Bondok et al., J. Anat. 160:161-174 (1988); and Cornett et al., J. Histochem. And Cytochem. 28:462-464 (1980); and mitoxantrone disclosed in Vibet et al., Drug Metabolism and Disposition 35:822-828 (2007); each incorporated by reference.
Problems Associated With Previously Described Fluorochromes Targeted to Lysosomes and Lysosome-Like Bodies
The accumulation of cationic amphiphilic drugs, toxic agents and other basic compounds inside acidic subcellular organelles is referred to as lysosomotropism. While many drugs require the presence of a cationic moiety for intrinsic bioactivity, their accumulation into subcellular organelles can also lead to undesirable tissue distribution, alkalinization of lysosomes, phospholipidosis and aberrant pharmacokinetic disposition (Ikeda et al, 2008). Over fifty cationic amphiphilic drugs, including antibiotics, antidepressants, antipsychotics, and antimalarial and antiarrhythmic agents are known to trigger phospholipidosis, which is typified by the excessive intracellular accumulation of phospholipids within lysosomes as lamellar bodies (Anderson and Borlak, 2006). Some commonly prescribed drugs known to perturb lysosomes by inducing phospholipidosis include propranolol, triparanol, chlorpromazine, chloroquine, fluoxetine, clindamycin and ketoconazole.
The origins of drug-induced lamellar bodies remain unresolved, though they appear to be generated by autophagic or heterophagic processes (Anderson and Borlak, 2006). Several mechanisms have been proposed to explain drug-induced phospholipidosis including the formation of drug-phospholipid complexes that are resistant to degradation by lysosomal phospholipases, direct inhibition of phospholipases themselves, and inhibition of intracellular pathways regulating phospholipid metabolism.
Only one instance can be found wherein a cationic amphiphilic fluorophore has been employed for monitoring drug-induced phospholipidosis (Lemieux et al, 2004). Lysotracker Red DND-99 (Invitrogen) was used to stain lysosomes and inhibition or displacement of the accumulated dye by test agents was subsequently monitored by fluorescence microscopy. Thus, the drug-induced vacuoles were not explicitly stained using the fluorescent probe, but rather an absence of signal was used as an indirect indication that the vacuoles were formed.
Numerous other in vitro methods for detecting phospholipidosis using different cell types including primary hepatocytes, peripheral blood monocytes and various cells lines, in combination with electron microscopy, flow cytometry, fluorescent microscopy or spectrofluorometry have previously been described (Ulrich et al, 1991; Cramer and Ulrich, 1994; Kasahara et al, 2006; Ikeda et al 2008; Gum et al, 2001; Lemieux et al 2004; Fujimura et al 2007; Tomizawa et al, 2006; Natalie et al, 2009; Nioi et al, 2008; Nioi et al, 2007; Bhandari et al, 2008; Morelli et al, 2006; Casartelli et al, 2003). Due to their inherent low throughput, however, many of these in vitro methods are not ideal for routine compound screening during early stage drug development. For example, electron microscopy, fluorescence microscopy and flow cytometry are generally considered low throughput platforms for routine drug screening purposes, especially when contrasted with fluorescence-based multi-well microplate assays. Additionally, some of the above cited assays depend upon difficult to procure cells. For instance, the preparation of primary hepatocytes or peripheral blood monocytes is too laborious for routine drug screening assay programs.
Many of the previously described cell-based methods require a relatively long period of treatment with the test agent before phospholipidosis can be detected (24-72 h). Additionally, the vast majority of the assays are based upon fluorescently labeled phospholipid analogs, such as NBD-PE, NBD-PC and Lipid-Tox reagent (Invitrogen, Carlsbad, Calif.), which require co-incubation of the analog with the drug during the extended incubation period. Distinct types of phospholipids appear to accumulate differently during phospholipidosis, however, and no explicit comparison of the performance of the different fluorescent lipid analogs has been performed to date (Diez-Blanco et al., 1987). The methods also depend upon permeabilization and fixation of the cells prior to analysis, which adds considerably to the overhead associated with the workfklow. What is needed is a fluorescent probe that faithfully highlights increases in vacuolar number and volume associated with exposure of cells and/or tissues to xenobiotic compounds.